Dopamine receptors Dopamine plays a major role in the regulation of cognitive, emotional and behavioral functions abnormalities in its regulation have been implicated in neuropsychiatric and substance use disorders. That dopamine D3 receptor (D3R) expression is elevated in response to drugs of abuse, has prompted efforts toward the development of D3R-selective agents for the treatment of drug addiction. Inhibition of D3R may be less prone to causing motor side effects that can result from D2R blockade. In addition to inhibiting the behavioral effects of cocaine, D3R partial agonists may lead to better compliance in treating addiction, given their ability to maintain some dopaminergic signaling tone rather than the complete blockade by an antagonist. Although some progress has been made in the development of selective ligands for D2R, D3R, or D4R, the receptor structural basis for this binding specificity is poorly understood and little insight into targeted efficacy design has been gained in the D2-like receptors. Even with high-resolution structural information, the high homology among the D2R-like receptors continues to provide a challenge to creating subtype-selective agents suitable for in vivo characterization and for clinical translation. By analyzing a series of D3R-selective compounds, which are characterized by 4-phenylpiperazine (primary pharmacophore, Ppharm) separated from an extended arylamide (secondary pharmacophore, Spharm) by a 4-carbon linking chain, we identified a divergent secondary binding pocket (SBP) that accommodates the Spharm of the compounds differently in D3R and D2R. Remarkably, experimental studies validated our predictions by identifying a single divergent Gly in the extracellular loop 1 as critical to D3R over D2R selectivity. These exciting results support our approach focusing on receptor dynamics. Thus, the subtype selectivity contributed by the SBP arises from particular non-conserved residues, their impact on the configuration and the dynamics of conserved residues, and thereby the overall size and shape of the SBP. We found that substituents on the phenyl ring and on the protonated nitrogen lead to distinct binding modes of the Ppharm in the OBS, and this correlates with the drastically different efficacies between different Ppharms. These findings establish the validity of our combined computational and experimental approach and form the basis for this project. Thus, our ligand design efforts center on the heretofore unexplored secondary cavities of the receptors. These cavities, however, do not necessarily form in the crystal structures but rather in conformational states that can only be addressed by advanced computational approaches exploring molecular dynamics. In addition, to understand the structural basis for the Na+-sensitivity of ligand binding to dopamine D2-like receptors, using computational analysis in combination with binding assays, we identified interactions critical in propagating the impact of Na+ on receptor conformations and on the ligand-binding site. Our findings expand the pharmacologically-relevant conformational spectrum of these receptors. Dopamine transporter DAT belongs to the Neurotransmitter:Sodium Symporter (NSS) family, and serves to terminate dopamine neurotransmission by recycling released dopamine back into the presynaptic neuron using the electro-chemical energy from the transmembrane Na+ gradient. DAT is the primary molecular target for abused psychostimulants such as cocaine and methamphetamine. Based on a wealth of information regarding the functional properties of NSSs, the crystal structures of LeuT, a bacterial NSS, reveal a central occluded substrate binding (S1) site in close association with two Na+ binding sites (the Na1 and Na2 sites), and an extracellular vestibule that binds inhibitors. Intriguingly, the configurations of these binding sites are significantly altered in various states. While these insights are critical, an understanding of the full spectrum of functional states and their transitions in a transporter cycle is required to understand the complexity of the binding modes and effects of ligands. Questions that have critical therapeutic implications are yet to be answered where and in which functional state the inhibitors bind and what their impact is on transport dynamics. The varied inhibition mechanisms of inhibitors are of particular interest in developing targeted and effective therapeutic interventions for drug abuse and other psychiatric disorders. Starting from LeuT structures, we were able to identify with computational simulations a second substrate binding (S2) site in the extracellular vestibule. In collaborative experiments we further found that the substrate in the S2 site triggers intracellular release of Na+ and substrate from the S1 site. Such results established our novel transport mechanism, which has been developed and validated further with experimental collaborators. Currently we focus on transitions among the distinct states, to reveal intermediate states to elucidate the dynamic nature of transport. Due to the lack of crystal structures of mammalian NSSs, we use the closely related LeuT and drosophila DAT as model systems to probe the mechanistic features shared within the NSS family. The demonstrations that cocaine, benztropines, and modafinil bind in the S1 site of DAT, are successes made possible by using our data from LeuT. From extensive MD simulations and free energy calculations performed under various conditions of Na+ and/or substrate binding and other functional elements, we identify intermediate states between the crystallographically-identified states in LeuT, and dissect the specific contribution of each element to global conformational transitions (GCTs). To understand the subtle but critical impact of the GCTs on the ligand binding modes, we investigate the identity of the allosteric interaction network (AIN), the configurational changes of which underlie GCTs. By simulating the impact of mutations or other factors known to disrupt GCTs, we use network and statistical correlation analysis to identify and connect the key structural motifs that form a coherent AIN, e.g., that connects the ligand binding region to the regions involved in the function. In particular, we have started to develop specific computational methodology to carry out comparative network analysis of distinct conformational states to assemble the AIN and identify the key nodes of the network. Insights into intermediate states, and the identity and dynamics of the AIN have been experimentally validated in both LeuT and DAT by our collaborators under conditions corresponding to those in the in silico studies.